The goal of this project is to systematically delineate the effects of chronic intermittent ethanol (CIE) exposure on GABAergic neuroactive steroids and their regulation in limbic brain areas after stress challenge. Both neuronal and extracellular neuroactive steroid levels will be examined by immunohistochemistry as well as gas chromatography-mass spectroscopy (GC-MS) analysis of microdialysates and/or tissue specimens, allowing investigation of limbic brain regions for the first time. We propose to test the hypotheses that ethanol exposure dysregulates basal or stress-induced levels of these steroids and these changes are related to ethanol consumption. We will further explore genetic contributions to adaptations in neurosteroid levels or responses. The first aim will determine the effects of CIE on neuronal vs. extracellular 3a,5a-THP levels in limbic/reward brain areas, including the prefrontal cortex, nucleus accumbens, amygdala, and bed nucleus of the stria terminalis of C57BL/6J mice. Aim 2 will delineate the effect of acute stress challenge on neuronal vs. extracellular 3a,5a-THP in limbic brain areas and the effect of stress challenge following five cycles of CIE in C57BL/6J mice. Aim 3 will determine if basal or CIE-related neuroactive steroid levels in limbic/reward brain areas are correlated with ethanol preference drinking across BXD mouse strains. Aim 4 will extend and compare our studies on neuroactive steroid adaptations to CIE in mice to primates. We will delineate the effects of prolonged ethanol drinking on 3a,5a-THP levels in limbic brain areas of rhesus monkeys. Our results will be integrated with changes in neuronal function, neurochemistry, gene expression and ethanol drinking behavior studies by our collaborators under the same experimental conditions. These studies will elucidate the effects of ethanol on GABAergic neuroactive steroids in limbic regions of brain and provide novel insights into the role of basal and stress-induced neuroactive steroids in various allostatic adaptations to chronic intermittent ethanol exposure.